Liu Gen, Chen Zhi-Gang, Yang Li-Rong, Rong Yu-Xia, Wang Qin, Li Li, Lu Qian-Wei, Jiang Ming-Dong, Qi Hong-Yi
College of Pharmaceutical Sciences, College of Chinese Medicine, Southwest University, 2 Tiansheng Road, Beibei District, Chongqing, 400715, China.
Radiotherapy Department, Chongqing Ninth People's Hospital, Chongqing, China.
Chin Med. 2023 Sep 21;18(1):123. doi: 10.1186/s13020-023-00808-7.
Nuclear receptors NUR77 and NOR1 were identified as critical targets in acute myeloid leukemia (AML) therapy. Previously, we showed that Z-ligustilide (Z-LIG) selectively targeted AML by restoring NUR77 and NOR1. However, its downstream mechanisms are yet to be elucidated.
SRB staining assay was used to measure cell viability. Cell apoptosis, mitochondrial membrane potential and mitochondrial reactive oxygen species were analyzed using flow cytometry. The potential targets of Z-LIG in AML HL-60 cells were evaluated by RNA sequencing. Changes in RNA levels were measured using quantitative RT-qPCR and western blot analysis was used to detect the expression of proteins.
Z-LIG preferentially induced mitochondrial dysfunction in HL-60 cells compared with 293T cells. Furthermore, RNA sequencing revealed that mitochondrial transcription and translation might be potential Z-LIG targets inhibiting HL-60 cells. NUR77/NOR1 overexpression significantly reduced the mitochondrial ATP and mitochondrial membrane potential and increased mitochondrial reactive oxygen species in HL-60 cells but not in 293T cells. Moreover, Z-LIG induced mitochondrial dysfunction by restoring NUR77 and NOR1 in HL-60 cells. Compared with HL-60 cells, the apoptosis-inducing activities of NUR77/NOR1 and Z-LIG were significantly reduced in HL-60 ρ0 cells depleted in mitochondrial DNA (mt-DNA). Moreover, NUR77/NOR1 and Z-LIG downregulated mitochondrial transcription and translation related proteins in HL-60 cells. Notably, Z-LIG remarkably reduced mitochondrial ATP in primary AML cells and showed anti-AML activity in mouse models of human AML.
Collectively, our findings suggested that Z-LIG selectively induces mitochondrial dysfunction in AML HL-60 cells by restoring NUR77 and NOR1, a process associated with interference in mtDNA transcription.
核受体NUR77和NOR1被确定为急性髓系白血病（AML）治疗中的关键靶点。此前，我们发现Z-藁本内酯（Z-LIG）通过恢复NUR77和NOR1选择性靶向AML。然而，其下游机制尚待阐明。
采用SRB染色法检测细胞活力。使用流式细胞术分析细胞凋亡、线粒体膜电位和线粒体活性氧。通过RNA测序评估Z-LIG在AML HL-60细胞中的潜在靶点。使用定量RT-qPCR测量RNA水平的变化，并使用蛋白质免疫印迹分析检测蛋白质表达。
与293T细胞相比，Z-LIG优先诱导HL-60细胞中的线粒体功能障碍。此外，RNA测序显示线粒体转录和翻译可能是Z-LIG抑制HL-60细胞的潜在靶点。NUR77/NOR1过表达显著降低HL-60细胞中的线粒体ATP和线粒体膜电位，并增加线粒体活性氧，但在293T细胞中未出现此现象。此外，Z-LIG通过恢复HL-60细胞中的NUR77和NOR1诱导线粒体功能障碍。与HL-60细胞相比，线粒体DNA（mt-DNA）耗竭的HL-60 ρ0细胞中，NUR77/NOR1和Z-LIG的凋亡诱导活性显著降低。此外，NUR77/NOR1和Z-LIG下调HL-60细胞中线粒体转录和翻译相关蛋白。值得注意的是，Z-LIG显著降低原发性AML细胞中的线粒体ATP，并在人AML小鼠模型中显示出抗AML活性。
总体而言，我们的研究结果表明，Z-LIG通过恢复NUR77和NOR1选择性诱导AML HL-60细胞中的线粒体功能障碍，这一过程与干扰mtDNA转录有关。
